This invention relates to a device for separating a fluid component, such as plasma, from a biologic sample, such as blood, using microspheres and analyte specific labeling. This invention also relates to a device and method for quantitative determination of an amount of analyte present in biologic fluids. The invention further relates to a quantitative. assay method and device for measuring one or more analytes in a biologic fluid sample using a point-of-care assay method and device. The test results can be analyzed using a suitable analyzer and, optionally, the assay test results are transmitted by way of digital transmission systems to permit further evaluation of the data.
There are presently many examples of one step assays for measuring analytes in fluid. A common assay is the pregnancy test device which involves contacting a urine sample with a test pad, which urine moves by capillary flow along the bibulous chromatography strips whereby the presence of human chorionic gonadotropin (HCG) will be detected usually as shown by a coloured line because of the reaction between HCG and reagents in the bibulous chromatography strips. This is an example of a chromatographic assay.
U.S. Pat. No. 5,766,961 issued Jun. 16, 1998 and U.S. Pat. No. 5,770,460 issued Jun. 23, 1998 are both entitled xe2x80x9cOne-Step Lateral Flow Nonbibulous Assayxe2x80x9d. xe2x80x9cNonbibulous lateral flowxe2x80x9d refers to liquid flow in which all of the dissolved or dispersed components of a liquid, which are not permanently entrapped or filtered out, are carried at substantially equal rates and with relatively unimpaired flow laterally through a stabilized membrane. This is distinguished from preferential retention of one or more components as would occur, for example, in materials capable of absorbing or imbibing one or more components, as occurs in chromatographic configurations. In this one-step assay, a sample (which may contain the analyte of interest) is collected on the xe2x80x9csample receiving zonexe2x80x9d from which it flows to the xe2x80x9clabelling zonexe2x80x9d at which point it encounters a specific binding reagent for the analyte coupled to visible moieties (the xe2x80x9cassay labelxe2x80x9d), then flows to a xe2x80x9ccapture zonexe2x80x9d where the analyte bound to visible moieties is captured.
In U.S. Pat. No. 5,540,888 issued Jul. 30, 1996 and entitled xe2x80x9cLiquid Transfer Assay Devicesxe2x80x9d, the invention described is a device for biochemical diagnostic assays. It comprises two liquid flow channels of porous material which transfer liquid by capillary flow to a common site following simultaneous application of the liquid to the ends of the channels. The channels interconnect at a certain point and then both continue in an arrangement analogous to an electrical bridge circuit. By selecting the hydraulic resistances of the arms of this circuit, the flow can be controlled across the bridge.
U.S. Pat. No. 5,300,779 issued Apr. 5, 1994 entitled xe2x80x9cCapillary Flow Devicexe2x80x9d describes methods and devices for measuring an analyte in a sample mixed with reagents, the devices defining a flow path. The specific binding by agglutination may provide for changes in flow rate, light patterns of a flowing medium, or light absorption or scattering which permit measurement of the analyte of interest.
In U.S. Pat. No. 5,110,724 issued May 5, 1992, entitled xe2x80x9cMulti-Analyte Devicexe2x80x9d, the invention described is an assay device for assaying multiple analytes in a drop-sized blood sample. A dispenser distributes a small volume blood sample to multiple transfer sites by capillary flow of the blood sample through sieving and distributing matrices which separate blood cells from plasma as the sample fluid migrates toward the transfer sites. A test plate in the device carries multiple absorbent pads, each containing reagent components for use in detection of a selected analyte. The test plate is mounted on the dispenser toward and away from a transfer position at which the exposed surface regions of the pads are in contact with associated sample-transfer sites, for simultaneous transfer of sample fluid from such sites to the pads in the support.
In U.S. Pat. No. 5,039,617 entitled xe2x80x9cCapillary Flow Device and Method for Measuring Activated Partial Thromboplastin Timexe2x80x9d, the invention described measures xe2x80x9cactivated partial thromboplastin timexe2x80x9d (APTT) on a whole blood sample by applying the sample to a capillary tract with reagents capable of initiating an APTT analysis, wherein clotting time is measured by the cessation of blood flow in the capillary tract. This is an example of a risk evaluation based on coagulation.
In U.S. Pat. No. 4,753,776 entitled xe2x80x9cBlood Separation Device Comprising a Filter and a Capillary Flow Pathway Exiting the Filterxe2x80x9d, the invention describes a method for separating plasma from red blood cells. The driving force for the movement of plasma from the filter to the reaction area of a device utilizing the method is capillary force provided by a tubular capillary. A filter is selected from glass microfiber filters of specified characteristics.
The U.S. Pat. No. 5,135,719 issued Aug. 4, 1992, entitled xe2x80x9cBlood Separation Device Comprising a Filter and Capillary Flow Pathway Exiting the Filterxe2x80x9d, the similar invention is described and the glass fibre filters are prepared from fibers with diameters between 0.10 and 7.0 xcexcm.
In U.S. Pat. No. 4,447,546 issued May 8, 1984, entitled xe2x80x9cFluorescent Immunoassay Employing Optical Fibre in Capillary Tubexe2x80x9d, a short length of precise diameter capillary tubing with an axially disposed optical fibre to which is immobilized a monolayer of a component of the antibody antigen complex (eg. an antibody) is described. The tubing is immersed in the sample.
U.S. Pat. No. 5,610,077 issued Mar. 11, 1997, entitled xe2x80x9cProcesses and Apparatus for Carrying Out Specific Binding Assaysxe2x80x9d, describes the well known antibody binding to antigen assay. The sample which may contain the analyte (a), (the substance being tested for) is mixed with (b) an antibody which binds to the substance being tested for, which antibody is immobilized on a solid support, and (c) another antibody for the substance being tested for which is conjugated to a detectable marker, to thereby form a complex between (b), the substance being tested for and (c) and causes the marker to be immobilized and detected.
In U.S. Pat. No. 4,943,522 issued Jul. 24, 1990, entitled xe2x80x9cLateral Flow, Non-Bibulous Membrane Assay Protocolsxe2x80x9d, the described invention is a method and apparatus for conducting specific binding pair assays, such as immunoassays, the test substrate is a porous membrane on which a member of the binding pair is affixed in an xe2x80x9cindicator zonexe2x80x9d. The sample is applied and is permitted to flow laterally through the indicator zone and any analyte in the sample is complexed with the affixed specific binding member, and detected. A novel method of detection employs entrapment of observable particle in the complex, for instance, red blood cells of blood can be used as the observable particles for detection of the complex.
An example of a method to separate red blood cells from whole blood samples is found in U.S. Pat. No. 5,118,428 issued Jun. 2, 1992, entitled xe2x80x9cMethod to Remove Red Blood Cells from Whole Blood Samplesxe2x80x9d. In the described invention, red blood cells are removed from whole blood samples with a solution containing an acid. The agglutinated red blood cells are then removed from the resulting suspension by procedures of filtration, centrifugation or decantation, leaving an essentially red blood cell-free serum or plasma sample.
In U.S. Pat. No. 5,073,484, entitled xe2x80x9cQuantitative Analysis Apparatus and Methodxe2x80x9d, an analyte is measured along a liquid flow path which includes a number of reaction-containing reaction zones spaced apart along the flow path. Detector means are employed to detect analyte, reactant or predetermined product in the reaction zones, the number of zones in which detection occurs indicating the amount of analyte in the liquid.
In U.S. Pat. No. 5,536,470 issued Jul. 16, 1986, entitled xe2x80x9cTest Carrier for Determining an Analyte in Whole Bloodxe2x80x9d, red blood cells cannot gain access from the blood sample application side, to the detection side and on the detection side as a result of an analysis reaction, an optically detectable change occurs.
A serious deficiency in current one-step assays for the measurement and/or detection of an analyte is that they provide only qualitative results rather than quantitative results. That is to say that the presence or absence of the analyte may be determined but the actual amount or concentration of analyte present in the sample would still not be known. The assay of the present invention provides quantitative results as the test is performed in a determinable volume. In the prior art methods it is not possible to consistently identify the exact volume of the test sample in repeated testings since the fluids must wash through the test strips.
Prior art methods using chromatographic strips and fiberglass strips require larger initial volumes of the biologic fluid in order to mobilize the proteins and labels in the strips. This is particularly true when the biologic fluid is blood and the plasma must first be separated from the blood sample. An advantage of the device and method of the present application is that very small fluid samples can be used to measure one or more analytes. The assay method and device of the present invention is also advantageous because the test volume can be made constant and therefore repeated testings will yield quantitative data which can be directly compared between samples and within a sample.
It is an advantage of the present invention that the assay device and methodology allows for separation of the plasma from the whole blood during the assaying of a fluid sample. In other words it is not necessary to previously separate out the cellular component of the blood before assaying the sample. This is a significant advantage as it allows that the assay can be used at the point of patient care, for example, by the patient themself, at the patient""s bedside or in a doctor""s office. In a preferred embodiment of the present invention there is provided by the device and assay methodology of the present invention a generic point-of-care platform suitable for use in one or more diagnostic or prognostic assays performed on one or more fluid samples.
In accordance with an aspect of the present invention a method for separating out the fluid component of a biologic sample using microspheres is provided. In one embodiment, the biologic sample is placed in contact with a group of microspheres and the fluid component separates from the sample as the fluid portion flows through the microspheres, by capillary action.
In accordance with an aspect of the present invention a quantitative assay method and device are provided for measuring one or more analytes in a fluid sample using a point-of-care assay method and device. The assay and device are designed for use by a patient themself, at the bedside of a patient, or in a doctor""s office. The test results are analyzed using a suitable analyzer and, optionally, the assay test results are transmitted by way of digital transmission systems to permit further evaluation of the data by an off-site professional.
In accordance with an aspect of the present invention, an assay method and portable assay device are provided for testing small volumes of biologic fluids, including blood, in a timely manner. In accordance with another aspect of the present invention, a method and device are provided for testing samples of biologic fluids in which a consistent volume of the biologic fluid sample is tested for one or more analytes and the data generated from the tests are used for collecting and compiling in a database pertaining, for example, to a particular disease condition. Ultimately the data collected can be used to train neural network algorithms and the algorithms may then be used to provide diagnostic and/or prognostic information based on the individual test results of any given test subject.
In accordance with another aspect of the present invention in respect to the analysis of blood, the cellular components of blood are separated from plasma by allowing the whole blood to be exposed to microsphere beads which permit the plasma to pass in the spaces formed between the microspheres by capillary action but not the cellular component. The present invention is not limited to the separation of cells from plasma in blood but includes broader applications where microsphere beads may be used to separate a fluid component from a cellular component in a biologic fluid. The microsphere beads are effectively acting as a fluid filter.
According to another aspect of the present invention a device is provided for separating plasma from blood in a sample. The device comprises a plurality of microspheres disposed in abutting relation and forming therebetween a plurality of capillary channels, whereby when the microspheres are disposed in fluid communication with a blood sample cellular and plasma components of the biologic sample are separated by capillary flow of the plasma component through the capillary channels formed by the interstitial spacing between abutting microspheres.
According to another aspect of the present invention the device comprises a plurality of groups of smaller microspheres each impregnated with a different label and interspersed with the larger microspheres in separate zones of the larger microspheres. The microspheres may be of substantially the same diameter, or the microspheres may be of differing diameters. The size of microsphere selected may be based on the viscosity of the sample or the size of the component one wishes to exclude or separate.
In accordance with yet another aspect of the present invention, the microspheres are bundled in a fluid-permeable material or the microspheres are maintained in abutting relation by a surface tension of the fluid which passes through them, for example plasma. In accordance with yet another aspect of the present invention the microsphere beads, also known simply as microspheres, are dried on a surface of the device.
In accordance with another aspect of the present invention, the device comprises a sample shelf adjacent to the fluid entrance and the microspheres are disposed on the sample shelf.
According to yet another aspect of the present invention the device comprises a plurality of smaller microspheres which are impregnated with at least one label interspersed with a plurality of larger microspheres such that the smaller microspheres occupy the interstitial spacing between the larger microspheres and release a label into the fluid as it flows through the interstitial spacing between the larger microspheres. There may be a plurality of groups of smaller microspheres each impregnated with a different label and interspersed with the larger microspheres in separate zones of the larger microspheres. Alternatively, the smaller microspheres may be mobilized and carried forward by the fluid as it passes along the capillary channels formed by the larger microspheres.
In accordance with another aspect of the present invention, the device comprises an indicator containing patient identification information to be associated with results of the assay, for example a bar code which can be read by a bar code reader.
According to another aspect of the present invention, a method of separating fluid from a biologic sample is provided. The sample has a fluid component and a non-fluid component and the method comprises the steps of,
(a) bringing the sample into fluid communication with a plurality of microspheres disposed in abutting relation and forming therebetween a plurality interstitial spaces which connect to comprise capillary channels, and
(b) collecting the fluid component as it is separated by capillary flow of the fluid component through the capillary channels. According to another aspect of the present invention there is provided, a method of conducting an assay utilizing a device comprising a capillary chamber defined by first and second opposed surfaces spaced a capillary distance apart having a fluid entrance and at least one reagent disposed within the capillary chamber, comprising the steps of,
(a) conveying a fluid sample into fluid communication with the fluid entrance such that the fluid sample is drawn into the capillary chamber by capillary action and reacts with the reagent, and
(b) analyzing the reagent to determine whether the reagent binds to an analyte in the fluid sample.
According to another aspect of the present invention the method further comprise the step of analyzing the reagent to determine a proportion of the reagent which binds to the sample.
According to another aspect of the present invention, the method further comprises a plurality of capillary chambers for conducting a plurality of assays on one or more fluid samples. According to another aspect of the present invention the results of the tests are recorded in a computer database and may be further applied in a trained neural network algorithm to generate a profile of one or more selected disorders. The assay further comprising the step of applying a receiver operating characteristic analysis to the data to determine a statistical significance of the data.
In accordance with another aspect of the present invention a wick or a capillary is brought into fluid communication with the fluid sample to remove the fluid sample from the capillary chamber.
In accordance with another aspect of the present invention microspheres are used to separate a cellular component from a fluid component in a biologic fluid, for example plasma from whole blood, and the fluid component can be tested in chromatography test strips. Furthermore, the microsphere beads of the present invention may be used as a labeling device, in addition to a filtration device, in standard nitrocellulose chromatography assays.
Other and further details of this preferred embodiments are described in the Detailed Description of the Preferred Embodiments together with the drawings described below.